The fumdamental objective of the proposed research is to investigate the role of non-histone proteins in maintaining the structure of the HeLa genome through the cell cycle. To this end, polyacrylamide gel electrophoresis will be applied to compare in a quantitative manner the non-histone proteins that are associated with HeLa metaphase chromosomes, nuclei and chromatin preparations. This comparison will be made more precise through use of the technique of one-dimensional peptide mapping by limited proteolysis. In addition, two-dimensional gels (isoelectric focusing/SDS discontinuous) will be applied to characterize the non-histone proteins to a high resolution. With these two-dimensional gels, the synthesis and modifications (phosphorylations, acetylations, poly ADP-ribosylations etc.) of the HeLa proteiins of synchronized cells will be followed through the cell cycle. The major non-histone proteins which are implicated in maintaining the long-range organization of the HeLa genome will be purified and further characterized by various biochemical and physical chemical technqiues. For example, the amino acid composition and sequence, DNA binding capacity, and hydrodynamic properties could be determined. Metaphase chromosomes will be isolated from other cell types in order to compare the non-histones of the chromosomes and "scaffolds" with those from HeLa cells. It has recently been shown that when HeLa metaphase chrosomes are depleted of histones with solutions containing dextran sulfate or high salt, the particles still resemble expanded metaphase chromosomes. This residual structure is due to a number of non-histone proteins which constrain the DNA into loops. As part of this proposed research, the histone-depletion procedure will be extended to other stages of the cell cycle. Preliminary results have suggested that non-histone proteins help to maintain the organization of HeLa DNA in interphase nuclei. In addition, nuclei from defined states of the cell cycle will be treated with nuclease besides extracting the histones to produce structures analogous to the metaphase "scaffolds". Electron microscopy will be used to learn something of the organization of the DNA in histone-depleted nuclei.